This invention relates generally to immunoassays, and more particularly, relates to an immunoassay useful for detecting and differentiating antibodies to Human Immunodeficiency Virus Type 1 (HIV-1) group M, HIV-1 group O and Human Immunodeficiency Virus Type 2 (HIV-2) in test samples with a rapid turn-around time.
Currently, there are two major phylogenetic groups of HIV-1 designated as groups "M" and "O." G. Meyers et al., Human Retroviruses and AIDS 1995, Los Alamos National Laboratory, Los Alamos, N.Mex. (1995). HIV-1 group M isolates further have been divided into subgroups (A to J) that are phylogenetically approximately equidistant from each other. Group M isolates predominate worldwide. The earliest reports about the sequence of HIV-1 group O viruses indicated that these viruses were as closely related to a chimpanzee virus as to other HIV-1 subgroups. See, for example, L. G. Gurtler et al., J. Virology 68: 1581-1585 (1994); M. Vanden Haesevelde et al., J. Virology 68: 1586-1596 (1994); De Leys et al., J. Virology 64: 1207-1216 (1990); DeLeys et al., U.S. Pat. No. 5,304,466; L. G. Gurtler et al., European Patent Publication No. 0591914A2. The group O sequences are the most divergent of the HIV-1 sequences described to date. Although HIV-1 group O strains are endemic to west central Africa (Cameroon, Equatorial Guinea, Gabon, and Nigeria), patients infected with group O isolates now have been identified in Belgium, France, Germany, Spain and the United States. See, for example, R. DeLeys et al., supra; P. Charneau et al., Virology 205:247-253 (1994); I. Loussert-Ajaka et al., J. Virology 69:5640-5649 (1995); H. Hampl et al., Infection 23:369-370 (1995); A. Mas et al., AIDS Res. Hum. Retroviruses 12:1647-1649 (1996); M. A. Rayfield et al., Emerging Infectious Diseases 2:209-212 (1996), and M. Peeters et al., AIDS 11:493-498 (1997).
HIV-1 group M serology is characterized in large part by the amino acid sequences of the expressed viral proteins (antigens), particularly those comprising the core and envelope (env) regions. These antigens are structurally and functionally similar, but have divergent amino acid sequences that elicit antibody responses which are specific for the particular antigen.
One of the key serological targets for detection of HIV-1 infection is the 41,000 molecular weight transmembrane protein (TMP), glycoprotein (gp)41. gp41 is a highly immunogenic protein which elicits a strong and sustained antibody response in individuals considered seropositive for HIV. Antibodies to this protein are among the first to appear at seroconversion. The immune response to gp41 apparently remains relatively strong throughout the course of the disease, as evidenced by the near universal presence of anti-gp41 antibodies in asymptomatic as well as clinical stages of AIDS. A significant proportion of the antibody response to gp41 is directed toward a well-characterized immunodominant region (IDR) within gp41.
HIV-2 infections have been identified in humans outside of the initial endemic area of West Africa, and have been reported in Europeans who have lived in West Africa or those who have had sexual relations with individuals from this region, homosexuals with sexual partners from the endemic area, and others. Cases of AIDS due to HIV Type 2 (HIV-2) now have been documented world-wide. See, for example, A. G. Saimot et al., Lancet i:688 (1987); M. A. Rey et al., Lancet i:388-389 (1987); A. Werner et al., Lancet i:868-869 (1987); G. Brucker et al., Lancet i:223 (1987); K. Marquart et al., AIDS 2:141 (1988); CDC, MMWR 37:33-35 (1987); Anonymous, Nature 332:295 (1988).
Serologic studies indicate that while HIV-1 and HIV-2 share multiple common epitopes in their core antigens, the envelope glycoproteins of these two viruses are much less cross-reactive. F. Clavel, AIDS 1:135-140 (1987). This limited cross-reactivity of the envelope antigens is believed to explain why currently available serologic assays for HIV-1 may fail to react with certain sera from individuals with antibody to HIV-2. F. Denis et al., J. Clin. Micro. 26:1000-1004 (1988). Recently issued U.S. Pat. No. 5,055,391 maps the HIV-2 genome and provides assays to detect the virus.
Concerns have arisen regarding the capability of currently available immunoassays for the detection of antibody to HIV-1 (group M) and/or HIV-2 to detect the presence of antibody to HIV-1 group O. I. Loussert-Ajaka et al., Lancet 343:1393-1394 (1994); C. A. Schable et al., Lancet 344:1333-1334 (1994); L. Gurtler et al., J. Virol. Methods 51:177-184 (1995). Compounding the problem of analyzing whether these immunoassays are capable of detecting group O is the limited availability of sera samples from patients who are infected with and/or have antibody to HIV-1 group O isolates. To date, few patients have been diagnosed with infection to HIV-1 group O isolates outside of west Central Africa, leading researchers to screen patients in west central African countries for the virus. Screening procedures in west central Africa have been hampered both by the time necessary to perform these assays as well as the equipment required to do so. Conventional binding assays available for detecting antibodies to HIV-1 group M, HIV-1 group O and HIV-2 usually take about two to four or more hours to reach a result. These assays further involve utilizing equipment including incubators and label reading devices that require electricity in order to operate. These assays incorporate specific binding members, usually antibody and antigen immunoreactants, wherein one member of the specific binding pair is labeled with a signal-generating compound (e.g., an antibody labeled with an enzyme, a fluorescent compound, a chemiluminescent compound, a radioactive isotope, a direct visual label, etc.). The test sample suspected of containing the analyte can be mixed with a labeled reagent, e.g., labeled anti-analyte antibody, and incubated for a time and under conditions sufficient for the immunoreaction to occur. The reaction mixture is subsequently analyzed to detect either that label which is associated with the analyte/labeled reagent complex (bound labeled reagent) or that label which is not complexed with analyte (free labeled reagent). The presence and/or amount of an analyte is indicated by the analyte's capacity to bind to a labeled reagent and binding member, which usually is immobilized or an insoluble complementary binding member.
There are situations and places in which the period of time usually required to perform these assays and report results is too long (i.e., two to four hours), or the equipment and/or electricity necessary to run the assay is not available. In such situations, a preferable test should be inexpensive, require little or no equipment, and provide a result for a screening assay in as little time as five minutes.
The use of reagent-impregnated teststrips in specific binding assays is well-known. See, for example, Deutsch et al., U.S. Pat. No. 4,361,537 and Brown et al., U.S. Pat. No. 5,160,701. In such procedures, a test sample is applied to one portion of the teststrip and is allowed to migrate or wick through the strip material. Thus, the analyte to be detected or measured passes through or along the material, possibly with the aid of an eluting solvent which can be the test sample itself or a separately added solution. The analyte migrates into or through a capture or detection zone on the teststrip, wherein a complementary binding member to the analyte is immobilized. The extent to which the analyte becomes bound in the detection zone can be determined with the aid of the labeled reagent which also can be incorporated into the teststrip or which can be applied separately.
In general, teststrips involve a material capable of transporting a solution by capillary action, i.e., a wicking or chromatographic action as exemplified in Gordon et al., U.S. Pat. No. 4,956,302. Different areas or zones in the teststrip contain the assay reagents needed to produce a detectable signal as the analyte is transported to or through such zones. The device is suitable both for chemical assays and binding assays and uses a developer solution to transport analyte along the strip. Also, to verify the stability and the efficacy of the assay reagents needed to produce the detectable signal, existing assays typically require at least that one or more strips from each manufacturing lot be separately assayed for both positive and negative controls.
Assay systems developed for the separate or concurrent detection of antibodies to HIV-1 group M, and/or HIV-1 group O and/or HIV-2 therefore must contain reagents which are useful for determining the specific presence of antibody to any or all of the viruses in a test sample while differentiating between them. The need therefore exists for reagents capable of reacting only with antibody to HIV group M, HIV group O and HIV-2, which reagents either exhibit no cross-reactivity or limited cross-reactivity with each other. It also would be beneficial to provide a disposable assay device which could incorporate these reagents and be used for screening individuals and providing results in a short amount of time.